Transcriptional regulation in prokaryotic systems will be studied in vitro at the level of E. coli RNA polymerase - DNA interactions. Physical and biochemical techniques will be used to identify and characterize the step(s) involved in polymerase binding and initiation that are most sensitive to promoter sequence and thereby determine promoter strength. Abortive initiation and a new fluorescence technique that directly measures polymerase binding to templates containing s4T will be used along with more standard procedures. The spectroscopic probe is particularly suited to measuring weak interactions in solution. Initial experiments will be done with restriction fragments carrying strong and weak promoters of T7 and lambda and with promoters such as lac that require positive control factors. I will also begin experiments using RNA polymerase I and the extrachromosomal ribosomal RNA genes of Tetrahymena to develop a simple in vitro system for study of selective transcription in eukaryotes. A basic understanding of how protein-nucleic acid interactions in general control gene expression will contribute to the eventual understanding of normal cell differeniation and of abnormal cell growth in humans.